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Crude alkaloid extract of Rhazya stricta inhibits cell growth and sensitizes human lung cancer cells to cisplatin through induction of apoptosis - PubMed

Crude alkaloid extract of Rhazya stricta inhibits cell growth and sensitizes human lung cancer cells to cisplatin through induction of apoptosis

Ayman I Elkady. Genet Mol Biol. 2013 Mar.

Abstract

There is an urgent need to improve the clinical management of non-small cell lung cancer (NSCLC), one of the most frequent causes of cancer-related deaths in men and women worldwide. Rhazya stricta, an important medicinal plant used in traditional Oriental medicine, possesses anti-oxidant, anti-carcinogenic and free radical scavenging properties. This study was done to explore the potential anticancer activity of a crude alkaloid extract of R. stricta (CAERS) against the NSCLC line A549. CAERS markedly suppressed the growth of A549 cells and considerably enhanced the anti-proliferative potential of cisplatin. CAERS-mediated inhibition of A549 cell growth correlated with the induction of apoptosis that was accompanied by numerous morphological changes, DNA fragmentation, an increase in the Bax/Bcl-2 ratio, the release of mitochondrial cytochrome c, activation of caspases 3 and 9 and cleavage of poly(ADP-ribose)-polymerase. CAERS reduced the constitutive expression of anti-apoptotic proteins (Bcl-2, Bcl-XL, Mcl-1 and Survivin) and cell cycle regulating proteins (cyclin D1 and c-Myc), but enhanced expression of the proapoptotic proteins Noxa and BAD. These observations indicate that CAERS induced apoptosis and sensitized NSCLC to cisplatin via a mitochondria-mediated apoptotic pathway. These data provide a rationale for using a combination of CAERS and CDDP to treat NSCLC and other CDDP-resistant tumors.

Keywords: RT-PCR; apoptosis; lung cancer; medicinal plant; western blot.

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Figures

Figure 1
Figure 1

CAERS suppresses cell growth and colony formation and sensitizes A549 cells to CDDP. A549 (A) and HF-5 (B) cells (50 × 103 cells/well) were seeded onto 24-well plates and treated with the indicated concentrations of CAERS alone for 24 h (dotted line), 48 h (solid line) and 72 h (dashed line). (B and C) A549 cells (50 × 103 cells/well) were seeded onto 24-well plates and treated with the indicated concentrations of CDDP alone (C) or CDDP + CAERS (D) for 24, 48 and 72 h. The inhibition of cell proliferation was assessed by the trypan blue dye exclusion assay. Cell viability was expressed as a percentage of control (no drug treatment) cell viability. The points are the mean ± SD of at least three independent experiments. (E) Effects of CAERS on clonogenicity of A549 cells. Cells were seeded onto a 6-well plate at 1000 cells/well and treated with the indicated concentrations of CAERS as detailed in Materials and Methods. The colonies were counted under a dissection microscope. A survival of 100% corresponded to the number of colonies obtained with cells that were not treated with CAERS. The experiment was repeated three times and the colony forming potential of the cells at each concentration of CAERS is expressed as a percent of the control and is reported as the mean ± SD. *p < 0.05 and #p < 0.001 compared to control cells (100%).

Figure 2
Figure 2

CAERS induces apoptosis in A549 cells. (A) Morphological changes in A549 cells after incubation with CAERS. (A) The cells were treated with the indicated concentrations of CAERS for 24 h after which they were examined using an inverted microscope (first row: magnification − 200x; second row: magnification − 400x). (B) The cells were stained with ethidium bromide for 20 min and photographed with a fluorescence microscope using a red filter (magnification: 200x). (C) Fragmentation of DNA isolated from A549 cells incubated with CAERS. The cells were incubated with the indicated concentrations of CAERS for 24 h after which DNA fragmentation was assessed as described in Materials and Methods. Lane `M’ indicates the DNA marker ladder. The data are representative of three independent experiments with similar results.

Figure 3
Figure 3

CAERS modulates expression of apoptosis regulatory proteins and their activation in A549 cells. A549 cells (20 × 104 cells/well) were seeded onto 6-well plates and treated with the indicated concentrations of CAERS for 24 h. Subsequently, 20 μg of cell extract protein isolated from CAERS-treated A549 cells was subjected to SDS-PAGE in 10% polyacrylamide gels, transferred to PVDF membranes and immunoblotted with antibodies against the proteins of interest. Treatment with CAERS increased the Bax/Bcl-2 ratio (A), promoted mitochondrial cyto c release (B) and activated caspases 9 and 3 and PARP cleavage (C). The immunoblots of cyto c (B) were stripped and re-probed with anti-cytochrome oxidase IV (COXIV) to confirm the purity of the cytoplasmic fraction and equal loading of the mitochondrial fraction. In all experiments, the membranes were stripped and re-probed with anti-β-actin antibody as a loading control. The data are representative of three separate experiments.

Figure 4
Figure 4

CAERS modulates the expression of antiapoptotic and cell cycle -regulated gene products in A549 cells. A549 cells (20 × 104 cells/well) were seeded onto 6-well plates and treated with the indicated concentrations of CAERS for 24 h. Total RNA was then isolated, reverse transcribed and subjected to PCR with gene-specific primers. The PCR products of the genes were then subjected to electrophoresis in 1% agarose gels and visualized by staining with ethidium bromide. HPRT1 was used as the internal control. A typical result from three independent experiments is shown.

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